The efficient delivery of therapeutic genes to non-dividing cells with resultant long-term stable expression remains a major goal in the development of gene therapy. None of the currently available vector systems has been shown to be capable of both efficient transduction of non-dividing cells and long term expression through stable integration of the vector genome into host cell DNA. Retroviral vectors based on Moloney murine leukemia virus, while capable of integration and stable long term expression, require cell division for efficient transduction. Miller D. G. et al., Mol. Cell. Biol. 10, 4239-4242 (1990). In contrast, vectors based on adenovirus and herpes simplex virus are capable of transducing non-dividing cells but do not integrate into host cell DNA with any appreciable frequency. See Grunhaus A. et al., Seminars in Virol. 3, 237-252 (1992); Geller A. I. et al., Proc. Natl. Acad. Sci. U.S.A. 87, 1194-1153 (1990). The less well characterized vectors based on the dependent parvovirus, adeno-associated virus (AAV), have been shown to integrate but their potential for transducing non-dividing cells has yet to be fully investigated. See Hermonat P. L. et al., Proc. Natl. Acad. Sci. U.S.A. 81, 6466-6470 (1984); Lebrowski J. S. et al., Mol. Cell. Biol. 8, 3988-3996 (1988); McLaughlin S. K. et al., J. Virol. 62, 1963-1973 (1988); and Samulski R. J. et al., J. Virol. 63, 3822-3828 (1989).
Adeno-associated virus (AAV) vectors are among a small number of recombinant virus vector systems which have been shown to have utility as both in vitro and in vivo gene transfer vectors (reviewed in Carter, 1992, Curr. Opinion Biotech. 3, 533-539 (1992); Muzcyzka, Curr. Top. Microbiol. Immunol. 158, 97-129) and thus are potentially of great importance for human gene therapy. AAV vectors are capable of stable DNA integration and expression in a variety of cells including cystic fibrosis (CF) bronchial and nasal epithelial cells (Flotte et al., Am. J. Respir. Cell Mol. Biol. 7, 349-356 (1992a)); Egan et al., Nature, 358, 581-584 (1992); Flotte et al., J. Biol. Chem. 268, 3781-3790 (1993a); Flotte et al., Proc. Natl. Aced. Sci. U.S.A. 90, 1613-1617 (1993b), human bone marrow-derived erythroleukemia cells (Walsh et el., Proc. Natl. Aced. Sci. U.S.A. 89, 7257-7261 (1992)), and several others. See also Flotte et al., Proc. Natl. Acad. Sci. U.S.A., 90, 10613-10617 (1993).
We have recently demonstrated that AAV vectors preferentially transduce cells in S phase of the cell cycle. However, transduction events do occur independent of S phase at low frequency. The factors within an S phase cell that facilitate transduction by AAV vectors remain undefined but are likely to directly involve or be closely linked to DNA synthesis. Possible factors include host cell polymerases required for the conversion of the single-stranded input genomes to double-stranded molecules and/or cellular factors facilitating vector integration.